NORTH-WESTERN JOURNAL OF ZOOLOGY 7 (2): 236-242 ©NwjZ, Oradea, Romania, 2011 Article No.: 111129 www.herp-or.uv.ro/nwjz

Body size and age of the Wood ( chensinensis) in northeastern China

Bing Yao CHEN1,3, Wen Bo LIAO2,3,* and Zhi Ping MI2,3

1. College of Life Science, Nanjing Normal University, Nanjing, 210097, China. 2. Key Laboratory of Southwest China Wildlife Resources Conservation Ministry of Education, China West Normal University, Nanchong, 637009, China. 3. Institute of Rare and Plants, China West Normal University, Nanchong 637009, China. *Correspondence at: W.B. Liao, E-mail: [email protected].

Received: 03. January 2011 / Accepted: 28. July 2011 / Available online: 09. August 2011

Abstract. Age structure and body size of a population of the China Wood Frog (Rana chensinensis) in northeastern China were determined by using skeletochronology. Lines of arrested growth (LAGs) recorded in phalanges were used to estimate the age of adults. Results showed that average age did not differ significantly between males and females. Age at sexual maturity in both sexes was 1 yr. The maximum observed longevity was 5 yrs in males and 4 yrs in females, respectively. Average body size of females was significantly larger than males. A non-significant negative correlation between body size and air temperature was found among the seven populations when the effect of altitude, latitude and age was controlled, suggesting that latitudinal variation in body size of Rana chensinensis across different temperature environment do not follow Bergmann's rule.

Key words: skeletochronology, Bergmann's rule, Rana chensinensis, body size, age structure.

Introduction morphosed juveniles first emerge about 2 months after egg-laying (Lu et al. 2006). The body size and Bergmann's rule states that body size of endo- age and growth rate of this are strongly in- therms tend to be larger at high latitudes or high fluenced by ambient temperature across small ele- altitudes (Bergmann 1847). This trend holds for vation ranges in Shanxi province, northern China the intraspecific body size variation in birds and (Ma 1985, Lu et al. 2006, Ma et al. 2009a). mammals (Freckleton & Pagel 2003, Meiri & Skeletochronology is a reliable method of age Dayan 2003). Body size variation of ectotherms estimation by counting the lines of arrested has the same trend (Ray 1960). However, for ecto- growth (LAGs) recorded in long bones in am- therms such as , there is considerable phibians (Hemelaar & Van Gelder 1980) and, has controversy about the direction of latitudinal been successfully used in assessing age for desert clines at the intra- and interspecific level (Ashton zones (Sullivan & Fernandez 1999), tropical re- 2002, Laugen et al. 2005, Olalla-Tarraga & Rodri- gions (Khonsue et al. 2000, Kumbar & Pancharatna guez 2007, Adams & Church, 2008, Cvetkovic et 2001), subtropical regions (Morrison et al. 2004, al. 2009). In most anurans, age and body size of Lai et al. 2005, Liao & Lu 2010a,b,c, Liao et al. 2010, individuals at high altitudes or high latitudes Liao & Lu 2011a, Li et al. 2010) and temperate re- (cold temperature) being older and larger than at gions (Guarino et al. 2003, Guarino & Erismis low altitudes or low latitudes (i.e. warm tempera- 2008, Kyriakopoulou- Sklavounou et al. 2008, Ma ture; Ashton 2002, Morrison et al. 2004, Lai et al. et al. 2009b, Üzüm 2009, Sinsch et al. 2010). 2005, Lu et al. 2006, Olalla-Tarraga & Rodriguez Here, we carried out a study for body size and 2007, Liao & Lu 2010b,c, Liao et al. 2011, Liao & Lu age in R. chensinensis using the data from a popu- 2011b). lation in northeastern China where these demo- The China Wood Frog (Rana chensinensis) is graphic parameters are unavailable. Moreover, we native to the montane regions of northern China used data on body size and age from six R. (Tanaka-Ueno et al. 1999). This frog is typically re- chensinensis populations based on the previous stricted to montane rivers, where it is the domi- studies (Lu et al. 2006, Ma et al. 2009a). Our aims nant anuran (Liu and Hu 1961, Xie et al. 2000). were to compare age, body size, and growth traits Rana chensinensis has a relatively long hibernating between the sexes in the population in northeast- period (3-5 months). Egg-laying lasts for 3-4 ern China and analyze variation in age, body size, weeks, and the frog may be classified as an explo- and growth among seven populations with differ- sive spring breeder (Wells 1977). Newly meta- ent temperature. Size and age of Rana chensinensis 237

Materials and Methods Kolmogorov–Smirnov test to identify age structure be- tween the sexes. Multifactorial analyses of covariance The fieldwork was conducted from January to February (ANCOVA) were used to assess the difference in body 2010 at Caohezhang Town (124º01′E, 41º04′N, and eleva- size between sexes when the effects of age were removed. tion 367 m) in Benxi County of Liaoning Province in The relationship between body size and age was analyzed northeastern China. The area has annual average tem- using one linear regression. We used partial correlation perature of 6-8°C (sub-zero mean monthly temperatures analysis to test variation in body size among populations occur in January–February) and annual total precipitation across latitudes with altitude and age as covariables. All of 700-1000 mm. The vegetation covering the study site is values given are shown as mean ± SD, and statistical tests characterized by tree species Xylosma racemosum, Fraxinus were two-tailed. mandshurica, Juglans mandshurica, Pinus koraiensis, Larix gmelinii. Individuals were captured by hand during the day in Results hollows under the riverbank, or in deep ponds at their hi- bernation sites. Each individual was sexed by the secon- A total of 123 individuals (63 males and 60 fe- dary sexual traits (the nuptial pads on their thumbs in males) were captured by chance in this population adult males and of the ova in adult females). The snout– vent length (SVL) was measured using a vernier caliper to resulting in a sex ratio of 1.05: 1. Lines of arrested the nearest 0.1 mm. The longest phalange of third toe of growth (LAGs) were present in cross sections of the right hind limb of each individual was removed by phalanges for 122 adult individuals of this species toe-clipping methods, and stored in 10% neutrally buff- (Fig. 1). The endosteal resorption of LAGs incom- ered formalin at room temperature for histological sec- pletely eroded the first (innermost) periosteal. tions. LAGs were observed in 28 males and 32 females. The standard skeletochronological procedure was However, the first LAGs of 6 males and 7 females used to determine age (e.g. Liao & Lu 2010a,b, Liao et al. 2010). For each individual digit, we cleaned off surround- were completely eroded by endosteal resorption ing tissues of the phalanges, and then put them in 5% ni- due to the absence of KL, and we added one year tric acid to decalcify for 48 h. These decalcified digits as their true age. For all samplings, endosteal re- were stained for 180 min in Harris’s haematoxylin. The sorption of LAGs did not affect LAGs counted. We stained bones were dehydrated by running the bone did not find double lines and false lines in any through successive ethanol stages of 70, 80, 95, and 100% samplings. for approximately 1 h in each concentration. Tissues were Average body size differed significantly be- infiltrated with successive paraffin changes for 1 h in an ± oven at 50 °C, embedded in small paraffin blocks and al- tween males and females (males, 60.6 2.9 mm; lowed to harden overnight. Cross-sections (13 μm thick- females, 67.7 ± 4.6 mm; Student’s t – tests: t = ness) of the phalanx with the smallest medullar cavity 10.17, p < 0.001). Within each age group, females and the thickest cortical bone were selected and mounted had significant larger body size than males (Table on glass slides. The sections were observed though a light 1). microscope and the best of them were photographed with a Motic BA300 digital camera mounted on a Moticam2006 Table 1. Body size (SVL, mm) within each age class of light microscope at ×400 magnification. The analysis of the China Wood Frog (Rana chensinensis) in northeast- lines of arrested growth (LAGs) was performed by two ern China. Values in descending order are mean ± SD persons (W.B. Liao & Z.P. Mi) with previous experience and sample size. of the technique and the results were then compared for age determination until consensus was reached. The in- Age class Females Males Z P nermost LAG can be reabsorbed with growth of endosteal 1y 66.1 ± 3.7 59.0 ± 2.5 3.62 < 0.001 tissue in amphibians which would affect the accuracy of (n = 8) (n = 13) age estimates (Smirina 1983). However, previous studies 2y 66.9 ± 3.5 60.0 ± 2.2 5.96 < 0.001 have shown that the endosteal resorption of LAGs occurs (n = 26) (n = 32) in primarily before they reach sexual maturity and 3y 69.0 ± 4.5 62.5 ± 3.4 3.34 0.001 slows down thereafter (Hemelaar 1985, Castanet et al. (n = 16) (n = 12) 1996, Liao & Lu 2010b, Liao et al. 2010b). Moreover, a 4y 69.0 ± 7.5 63.6 ± 1.9 2.07 0.04 weak reabsorption of LAGs in R. chensinensis was ob- (n = 9) (n = 5) served by noting the presence of the Kastschenko Line 5y 65.08 (KL; the interface between the endosteal and periosteal (n = 1) zones, Lu et al. 2006, Ma et al. 2009a) where many indi- viduals reach sexual maturity in one year. In this study, Age structure did not differ significantly be- we confirmed the endosteal resorption of LAGs in this tween the sexes (Fig. 2; Kolmogorov–Smirnov test: species based on the Kastschenko Line. Difference in body size and age between males and D = 0.32, p = 1.00). Males and females had same females was tested using Student’s t - tests. We applied sexual mature age of 1 yr. Longevity of males and 238 Chen, B.Y. et al.

Figure 1. Cross sections (A: a 1-yr old female; B: a 2-yr old male; C: a 3-yr old male; D: a 4-yr old female) of hema- toxylin-stained cross-sections of the phalangeal bone of adult China Wood Frog (Rana chensinensis) in north- eastern China. Arrows indicate the lines of arrested growth (LAG). KL represents Kastschenko Line, the inter- face between the endosteal and periosteal zones. Scale bar: 300 μm.

Figure 2. Adult age structure (male, open bars; female, close bars) of the China Wood Frog (Rana chensinensis) in northeastern China. Size and age of Rana chensinensis 239

females was 5 yrs and 4 yrs, respectively. For both 0.12, p = 0.82; Fig. 4), with individuals in low tem- sexes, dominant age was 2 yrs. There was signifi- perature not larger than those in high tempera- cant difference in average age between females ture. Average age is positively correlated with lati- and males (Student’s t – tests: t = 1.51, p = 0.13). tude in males (r = 0.80, p = 0.03) but not in females Average age of females was 2.4 ± 0.9 yrs, and that (r = 0.05, p = 0.91). of males was 2.2 ± 0.9 yrs. ANCOVA revealed that age significantly affect body size between males and females (F1, 121 = 15.64, p < 0.001). When the ef- Discussion fect of age was controlled, body size differed sig- nificantly between the sexes, with females being For R. chensinensis, no growth line formed in long also larger than males (ANCOVA: F1, 121 = 102.73, p bones of metamorphosed individuals was found, < 0.001). but a RL and LAGs were formed subsequently (Lu For the population, body size is positively cor- et al. 2006, Ma et al. 2009a). Although these lines related with age in males and females (One linear were partly destroyed by the resorption process, regression: males, SVL = 1.67 age + 56.98, F = we can confirm the final LAG of all individuals 23.04, r = 0.52, p < 0.001; females, SVL = 1.58 age + based on suggestions by Lu et al. (2006). 63.09, F = 3.25, r = 0.23, p = 0.08; Fig. 3). A non- Males reach sexual maturity one year earlier significant interaction between age and sex re- than females in most amphibians (Miaud et al. vealed that relationship between age and size did 1999, Kyriakopoulou-Sklavounou & Grumiro not differ apparently among the sexes (F1, 121 = 2002, Liao et al. 2010). However, the wood frogs 4.28, p = 0.07), suggesting that males and females from our results showed that sexual maturity of have similar growth pattern. males and females arose after the first hibernation. A non-significant relationship between aver- Age at sexual maturity in both sexes was 1 yr, age body size and latitude was found among the suggesting that individuals would spend more seven populations when removing the effect of al- time in reproduction. Longevity of females is titude and age (Partial correlations: males, r = 0.05, longer than that of males in most anurans (Khon- p = 0.94; females, r = 0.57, p = 0.32). There was also sue et al. 2001, Matthews & Miaud 2007). How- a non-significant correlation between body size ever, in the R. chensinensis population, males had and air temperature among these populations longer longevity than females. Ma (1985) reported when the effect of age was controlled (Partial cor- the maximum longevity was 5 yrs in male and fe- relations: males, r = 0.24, p = 0.65; females, r = - male. Our sample size is smaller than Ma (1985).

Figure 3. The relationship between age and body size (male, open bars; female, close bars) of the China Wood Frog (Rana chensinensis) in northeastern China. 240 Chen, B.Y. et al.

Figure 4. The relationship between adult size (Mean ± SD; male, open bars; female, closed bars) and air temperature of the China Wood Frog (Rana chensinensis) from the seven populations in north- ern China.

In most anuran species, there is a positive cor- growth rate between males and females, similar to relation between body size and age in both sexes the pattern for R. chensinensis reported by Lu et al. (Ryser 1996, Lu et al. 2006, Liao & Lu 2010a, Liao (2006). The ultimate mechanism behind size di- et al. 2010). However, our studies showed a posi- morphism may result from the difference in how tive and significant correlation in males but not in males and females allocate energy between females. Whereas other anurans with a positive growth and reproductive investment (Halliday & correlation may be true for only one sex (Gibbons Verrell 1988, Jørgensen 1992). & McCarthy 1984, Leclair & Castanet 1987, Cherry Body size variation in seven R. chensinensis & Francillon 1992). Body size ranges for different populations at different latitudes showed indi- age classes overlapped extensively, making age es- viduals from higher latitudes were not larger than timation from size data alone impossible for the those from lower latitude, supporting the idea that population. This is in agreement with previous amphibians do not follow Bergmann’s rule studies for the relationship between age and size (Ashton 2002, Adams & Church 2008, Liao et al. (Khonsue et al. 2000, Li et al. 2010, Liao & Lu 2010b). On the contrary, previous studies for ele- 2010b). vational variation in body size in this species from As in most anurans (Monnet & Cherry 2002), three different-altitudes along a montane river R. chensinensis females have larger body size than show that they exhibit a significant negative corre- males, showing a marked sexual size dimorphism. lation between body size and altitude consistent Numerous studies show that growth rates differ with Bergmann’s rule (Lu et al. 2006, Ma et al. between the sexes before attainment of maturity; 2009a). different age and/or body size between sexes at Age and growth rate affected variation in maturity, and different age structure between body size of adult amphibians. The relative con- sexes in breeding populations may explain sexual tributions of age and growth rate on body size dimorphism in anuran body size (Ryser 1988, across environmental conditions can confirm that Khonsue et al. 2001, Monnet & Cherry 2002). Re- some species conform to Bergmann’s clines while garding which factors predominantly affect size others reverse it (Ma et al. 2009b). Our findings dimorphism in the population from northeastern suggested that average body size in R. chensinensis China, ANCOVA revealed age as a major factor. did not significantly increase with decreasing In our study, growth rate might not affect sexual temperature. This is in disagreement with the differences in size, since there was a similar findings observed in R. chensinensis (Lu et al. 2006, Size and age of Rana chensinensis 241

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